Optical reader for diffraction grating-based encoded optical identification elements

ABSTRACT

An optical reader system  7  for diffraction grating-based encoded microbeads (or bead reader system), comprises a reader box  100,  which accepts a bead cell (or cuvette)  102  that holds the microbeads  8,  having an embedded code therein. The reader box  100  interfaces along lines  103  with a known computer system  104.  The reader box  100  interfaces with a stage position controller  112  and the controller  112  interfaces along a line  115  with the computer system  104  and a manual control device (or joy stick)  116  along a line  117.  The reader interrogates the microbeads to determine the embedded code and/or the fluorescence level on the beads. The reader provides information similar to a bead flow cytometer but in a planar format, i.e., a virtual cytometer.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplications, Ser. No. 60/512,302, filed Oct. 17, 2003; Ser. No.60/513,053, filed Oct. 21, 2003; Ser. No. 60/546,435, filed Feb. 19,2004; Ser. No. 60/610,829, filed Sep. 17, 2004; and is acontinuation-in-part of U.S. patent application Ser. No. 10/661,234filed Sep. 12, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/645,689, filed Aug. 20, 2003, which claimed thebenefit of U.S. provisional applications, Ser. No. 60/405,087 filed Aug.20, 2002 and Ser. No. 60/410,541, filed Sep. 12, 2002; and is acontinuation-in-part of U.S. patent application, Ser. No. 10/661,836,filed Sep. 12, 2003; all of which are incorporated herein by referencein their entirety.

The following cases contain subject matter related to that disclosedherein and are incorporated herein by reference in their entirety: U.S.patent application Ser. No. 10/661,234, filed Sep. 12, 2003, entitled“Diffraction Grating-Based Optical Identification Element”; U.S. patentapplication Ser. No. 10/661,031 filed Sep. 12, 2003, entitled“Diffraction Grating-Based Encoded Micro-particles for MultiplexedExperiments”; U.S. patent application Ser. No. 10/661,082, filed Sep.12, 2003, entitled “Method and Apparatus for Labeling Using DiffractionGrating-Based Encoded Optical Identification Elements”; U.S. patentapplication Ser. No. 10/661,115, filed Sep. 12, 2003, entitled “AssayStick”; U.S. patent application Ser. No. 10/661,836, filed Sep. 12,2003, entitled “Method and Apparatus for Aligning Microbeads in order toInterrogate the Same”; U.S. patent application Ser. No. 10/661,254 ,filed Sep. 12, 2003, entitled “Chemical Synthesis Using DiffractionGrating-based Encoded Optical Elements”; U.S. patent application Ser.No. 10/661,116, filed Sep. 12, 2003, entitled “Method of Manufacturingof a Diffraction grating-based identification Element”; and U.S. patentapplication Ser. No. 10/763,995, filed Jan. 22, 2004, entitled, “HybridRandom Bead/Chip Based Microarray”; and U.S. Provisional patentapplication, Ser. No. 60/555,449, filed Mar. 22, 2004, entitled,“Diffraction Grating-Based Encoded Micro-particles for MultiplexedExperiments”.

TECHNICAL FIELD

This invention relates to optical readers of optical identificationelements, and more particularly to an optical readers for diffractiongrating-based encoded optical identification elements.

BACKGROUND ART

A common class of experiments, known as a multiplexed assay ormultiplexed bio-chemical experiment, comprises mixing (or reacting) alabeled target analyte or sample (which may have known or unknownproperties or sequences) with a set of “probe” or reference substances(which also may have known or unknown properties or sequences).Multiplexing allows many properties of the target analyte to be probedor evaluated simultaneously (i.e., in parallel). For example, in a geneexpression assay, the “target” analyte, usually an unknown sequence ofDNA, is labeled with a fluorescent molecule to form the labeled analyte.One known type of assay is a “bead-based” assay where the probemolecules are attached to beads or particles.

For example, in a known DNA/genomic bead-based assay, each probeconsists of known DNA sequences of a predetermined length, which areattached to a labeled (or encoded) bead or particle. When a labeled“target” analyte (in this case, a DNA sequence) is mixed with theprobes, segments of the labeled target analyte will selectively bind tocomplementary segments of the DNA sequence of the known probe. The knownprobes are then spatially separated and examined for fluorescence. Thebeads that fluoresce indicate that the DNA sequence strands of thetarget analyte have attached or hybridized to the complementary DNA onthat bead. The DNA sequences in the target analyte can then bedetermined by knowing the complementary DNA (or cDNA) sequence of eachknown probe to which the labeled target is attached. In addition, thelevel of fluorescence is indicative of how many of the target moleculeshybridized (or attached) to the probe molecules for a given bead. As isknown, a similar bead-based assay may be performed with any set of knowand unknown molecules/analyte/ligand.

In such bead-based assays, the probes are allowed to mix without anyspecific spatial position, which is often called the “random bead assay”approach. In addition, the probes are attached to a bead so they arefree to move (usually in a liquid medium). Further, this approachrequires that each bead or probe be individually identifiable orencoded. In addition, a bead based assay has the known advantage thatthe analyte reaction can be performed in a liquid/solution byconventional wet-chemistry techniques, which gives the probes a betteropportunity to interact with the analyte than other assay techniques,such as a known planar microarray assay format.

There are many bead/substrate types that can be used for tagging orotherwise uniquely identifying individual beads with attached probes.Known methods include using polystyrene latex spheres that are coloredor fluorescent labeled. Other methods include using small plasticparticles with a conventional bar code applied, or a small containerhaving a solid support material and a radio-frequency (RF) tag. Suchexisting beads/substrates used for uniquely identifying the probes,however, may be large in size, have a limited number of identifiablecodes, and/or made of a material not suitable to harsh environmentalconditions, such as, harsh temperature, pressure, chemical, nuclearand/or electromagnetic environments.

Therefore, it would be desirable to provide encoded beads, particles orsubstrates for use in bead-based assays that are very small, capable ofproviding a large number of unique codes (e.g., greater than 1 millioncodes), and/or have codes which are resistant to harsh environments andto provide a reader for reading the code and/or the fluorescent labelattached to the beads.

Also, there are many industries and applications where it is desirableto uniquely label or identify items, such as large or small objects,plants, and/or animals for sorting, tracking, identification,verification, authentication, or for other purposes. Existingtechnologies, such as bar codes, electronic microchips/transponders,radio-frequency identification (RFID), and fluorescence (or otheroptical techniques), are often inadequate. For example, existingtechnologies may be too large for certain applications, may not provideenough different codes, cannot be made flexible or bendable, or cannotwithstand harsh environments, such as, harsh temperature, pressure,chemical, nuclear and/or electromagnetic environments.

Therefore, it would be desirable to obtain a labeling technique and/orencoded substrate for labeling items that provides the capability ofproviding many codes (e.g., greater than 1 million codes), that can bemade very small (depending on the application) and/or that can withstandharsh environments and to provide a reader for reading the code and/orthe fluorescent label attached to the beads.

SUMMARY OF THE INVENTION

Objects of the present invention include provision of a reader for anoptical identification elements where the elements may have a largenumber of distinct codes, may be made very small (depending on theapplication) and/or can withstand harsh environments.

According to the present invention, an optical reader for readingmicrobeads, comprises said reader capable of receiving at least onemicrobead disposed therein, each microbead having at least one codedisposed therein, said microbead having at least one diffraction gratingdisposed therein, said grating having at least one refractive indexpitch superimposed at a common location, said grating providing anoutput optical signal indicative of said code when illuminated by aninput light signal; a source light providing said input light signalincident at a location where said microbeads are located when loaded;and a reader which reads said output optical signal and provides a codesignal indicative of said code.

The present invention provides a reader for reading codes and/orfluorescence signals from an encoded optical identification elementscapable of having many different optically readable codes.

The reader of the present invention optimizes fluorescent measurementswhen microbeads having a cylindrical shape are used, while minimizingsensitivity to beam positioning and/or bead misalignment.

In addition, the invention can easily identify a bead and the codetherein along a scan having many beads along a row and compensates foruneven, jagged, and/or inconsistent surface geometries for the endeffects of the beads, as well as when beads densely packed end-to-end.

Further, because the code is projected and read in the “far field” orFourier plane, the reader of the present invention does not requireexpensive imaging and magnifying optics to create a high resolutionmagnified image of the bead to read the code. This is different fromprior readers which actually image the bead itself to determine thecode, e.g., for small particles that have bar codes printed on them.

The elements may be very small “microbeads” (or microelements ormicroparticles or encoded particles) for small applications (about1-1000 microns), or larger “macroelements” for larger applications(e.g., 1-1000 mm or much larger). The elements may also be referred toas encoded particles or encoded threads. Also, the element may beembedded within or part of a larger substrate or object.

The element has a substrate containing an optically readable compositediffraction grating having a resultant refractive index variation madeup of one or more collocated refractive index periods (or spacings orpitches A) that make-up a predetermined number of bits. The microbeadallows for a high number of uniquely identifiable codes (e.g.,thousands, millions, billions, or more). The codes may be digital binarycodes and are readable by the present invention.

The element may be made of a glass material, such as silica or otherglasses, or may be made of plastic or polymer, or any other materialcapable of having a diffraction grating disposed therein. Also, theelement may be cylindrical in shape or any other geometry, provided thedesign parameters are met. For certain applications, a cylindrical shapeis optimal. The gratings (or codes) are embedded inside (including on ornear the surface) of the substrate and may be permanent non-removablecodes that can operate in harsh environments (chemical, temperature,nuclear, electromagnetic, etc.).

The present invention reads the code in the element as well as anyfluorescence that may exist on the microbeads. In addition, theinvention may use the same laser to both interrogate the code and read afluorescent signal from the bead, without interference between the two,thereby saving cost and time.

The present invention interrogates the beads on a planar surface, e.g.,a groove plate. The invention may act as a “virtual cytometer”, whichprovides a series of code and fluorescence data from a series of beads,similar to a flow cytometer; however, with in the present invention thebeads are disposed on a planar substrate. The beads may be aligned byother than grooves if desired. Alternatively, the surface need not beplanar, e.g., it may have a cylindrical or other non-planar shape, suchas that described in U.S. Provisional Patent Application Nos. 60/609,583and 60/610,910, which are incorporated herein by reference in theirentirety. Also, the reader may be used with a classical flow cytometerconfiguration if desired, where beads are flowed by the reader head in afluid stream.

In addition to reading the bead code and/or fluorescence, the reader candetermine the precise location of each bead read in the bead cell, andcan then return to any given bead for further review and/or analysis ifdesired. This feature also allows the reader to be used as a bead“mapper”, i.e., to identify or map the exact location of each bead on aplanar surface. Also, the reader could use fluorescent “tracer” beadshaving a predetermined fluorescent signal, different from the otherbeads, which would allow the reader to map the locations of all thebeads based on the location of the tracer beads. Further, once thelocation of the beads in a cell are mapped, the bead cell can be used inanother reader or scanner for review and/or analysis. Other techniquesmay also be used to orient the reader to a predetermined calibration orstandard cell location from which all the beads may be mapped ifdesired.

The foregoing and other objects, features and advantages of the presentinvention will become more apparent in light of the following detaileddescription of exemplary embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of an optical reader system, in accordancewith the present invention.

FIG. 2 is a block diagram of the overall architecture of the opticalreader, in accordance with the present invention.

FIG. 3 is a block diagram of the opto-mechanical architecture for theoptical reader architecture, in accordance with the present invention.

FIG. 4 is an optical schematic of a laser block assembly, in accordancewith the present invention.

FIG. 4 a is an optical schematic of an alternative laser block assembly,in accordance with the present invention.

FIG. 5 is an optical schematic of mode matcher optics, in accordancewith the present invention.

FIGS. 5 aand 5 b are diagrams of various excitation beam shapes onbeads, in accordance with the present invention.

FIG. 6 is an optical schematic of code pickup, fluorescence pick-up andvision pick-up, in accordance with the present invention.

FIG. 6A is an more detailed optical schematic of code pickup optics ofFIG. 6, in accordance with the present invention.

FIG. 6B is a more detailed schematic of the fluorescence pick-up opticsof FIG. 6.

FIG. 7 is an optical schematic of a photo-multiplier tube (PMT)assembly, in accordance with the present invention.

FIG. 8 is a front perspective view of an optical reader, in accordancewith the present invention.

FIG. 9 is a top and front perspective view of the optical reader of FIG.8, in accordance with the present invention.

FIG. 10 is a back and top perspective view of the optical reader of FIG.8, in accordance with the present invention.

FIG. 11 is a perspective view of a slide holder and slide with grooves,in accordance with the present invention.

FIG. 12 is a perspective view of a laser block assembly of the opticalreader of FIG. 8, in accordance with the present invention.

FIG. 13 is a side cross-sectional perspective view of the laser blockassembly of FIG. 12, in accordance with the present invention.

FIG. 14 is a perspective view of a turning mirror assembly of theoptical reader of FIG. 8, in accordance with the present invention.

FIG. 15 is a side cross-sectional perspective view of the turning mirrorassembly of FIG. 14, in accordance with the present invention.

FIG. 16 is a perspective view of a fluorescent detection and lightillumination assembly and additional optics of the optical reader ofFIG. 8, in accordance with the present invention.

FIG. 17 is a rotated perspective view of a portion the fluorescentdetection and light illumination assembly of FIG. 16, in accordance withthe present invention.

FIG. 18 is a cross-sectional perspective view of the fluorescentdetection and light illumination assembly of FIG. 17, in accordance withthe present invention.

FIG. 19 is a perspective view of a photo-multiplier tube (PMT) assemblyof the optical reader of FIG. 8, in accordance with the presentinvention.

FIG. 20 is a side cross-sectional perspective view of thephoto-multiplier tube (PMT) assembly of FIG. 19, in accordance with thepresent invention.

FIG. 20A is a front cross-sectional perspective view of thephoto-multiplier tube (PMT) assembly of FIG. 19, in accordance with thepresent invention.

FIG. 21 is a perspective view of a beam splitter and edge detectionassembly of the optical reader of FIG. 8, in accordance with the presentinvention.

FIG. 22 is a rotated cross-sectional perspective view of the beamsplitter and edge detection assembly of FIG. 22, in accordance with thepresent invention.

FIG. 23 is a side view of an optical identification element, inaccordance with the present invention.

FIG. 24 is a top level optical schematic for reading a code in anoptical identification element, in accordance with the presentinvention.

FIG. 25 is an optical schematic for reading a code in an opticalidentification element, in accordance with the present invention.

FIG. 26 is an image of a code on a CCD camera from an opticalidentification element, in accordance with the present invention.

FIG. 27 is a graph showing an digital representation of bits in a codein an optical identification element, in accordance with the presentinvention.

FIG. 28 illustrations (a)-(c) show images of digital codes on a CCDcamera, in accordance with the present invention.

FIG. 29 illustrations (a)-(d) show graphs of different refractive indexpitches and a summation graph, in accordance with the present invention.

FIG. 30 illustrations (a)-(b) are graphs of reflection and transmissionwavelength spectrum for an optical identification element, in accordancewith the present invention.

FIG. 31 is side view of a blazed grating for an optical identificationelement, in accordance with the present invention.

FIG. 32 is a side view of an optical identification element having acoating, in accordance with the present invention.

FIG. 33 is a side view of an optical identification element having agrating across an entire dimension, in accordance with the presentinvention.

FIG. 34, illustrations (a)-(c), are perspective views of alternativeembodiments for an optical identification element, in accordance withthe present invention.

FIG. 35, illustrations (a)-(b), are perspective views of an opticalidentification element having multiple grating locations, in accordancewith the present invention.

FIG. 36, is a perspective view of an alternative embodiment for anoptical identification element, in accordance with the presentinvention.

FIG. 37 is a view an optical identification element having a pluralityof gratings located rotationally around the optical identificationelement, in accordance with the present invention.

FIG. 38 illustrations (a)-(e) show various geometries of an opticalidentification element that may have holes therein, in accordance withthe present invention.

FIG. 39 illustrations (a)-(c) show various geometries of an opticalidentification element that may have teeth thereon, in accordance withthe present invention.

FIG. 40 illustrations (a)-(c) show various geometries of an opticalidentification element, in accordance with the present invention.

FIG. 41 is a side view an optical identification element having areflective coating thereon, in accordance with the present invention.

FIG. 42 illustrations (a)-(b) are side views of an opticalidentification element polarized along an electric or magnetic field, inaccordance with the present invention.

FIG. 43 shows a bit format for a code in an optical identificationelement, in accordance with the present invention.

FIGS. 44 & 45 show the use of a lens as an imaging and Fourier transformdevice, in accordance with the present invention.

FIGS. 46-51 show various graphs relating to fluorescence level as itrelated to excitation beam position on the bead, in accordance with thepresent invention.

FIG. 52 is a block diagram of an alternative architecture embodiments ofa bead reader or mapper, in accordance with the present invention.

FIGS. 53-56 are optical diagrams of an alternative embodiments of a beadreader or mapper, in accordance with the present invention.

FIGS. 57 and 58 show alternatives for a bead cell, in accordance withthe present invention.

FIGS. 59-61 show optical images of a bead being scanned for a code, inaccordance with the present invention.

FIGS. 62-65 show graphs and drawings relating to bead code readingtolerances, in accordance with the present invention.

FIG. 66-67 shows two graphs of optical power at detectors used to locatethe bead and code window, in accordance with the present invention.

FIG. 68 shows a sample assay process chart which could use the reader,in accordance with the present invention.

FIGS. 69-71 show dynamic sample dynamic range data and readerthroughput, in accordance with the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Referring to FIG. 1, an optical reader system 7 for diffraction gratingbased encoded optical identification elements (such as microbeads),comprises a reader box 100, which accepts a bead cell (or holder orcuvette or chamber) 102 that holds and aligns the microbeads 8 whichhave embedded codes therein. The reader box 100 interfaces along lines103 with a known computer system 104 having a computer 106, a displaymonitor 108, and a keyboard. In addition, the reader box 100 interfacesalong lines 114 with an stage position controller 112 and the controller112 interfaces along a line 115 with the computer system 104 and amanual control device (or joy stick) 116 along a line 117.

The microbeads 8 are similar to or the same as those described inpending U.S. patent application Ser. No. 10/661,234, entitledDiffraction Grating Based Optical Identification Element, filed Sep. 12,2003, which is incorporated herein by reference in its entirety,discussed more hereinafter.

The bead cell 102 is similar to or the same as that described in pendingU.S. patent application Ser. No. 10/661,836, entitled “Method andApparatus for Aligning Microbeads in order to Interrogate the same”,filed Sep. 12, 2003, as well as U.S. patent applications Ser. No.10/763,995 and Provisional Patent Application Nos. 60/609,583 and60/610,910, which are all incorporated herein by reference in itsentirety, discussed more hereinafter.

Referring to FIG. 2, the reader box 100 comprises the bead cell 102,certain opto-mechanical elements 120 including a code camera, a edgetrigger diode which measures a portion of the light reflected off thebeads and provides signal indicative thereof to electronics discussedhereinafter, a green laser and red laser (for fluorescence excitation,and code reading), 2 photo-multiplier tubes to detect 2 fluorescentsignals from the beads, a laser power diode to detect and/or calibratelaser power, an alignment/imaging light to illuminate the bead holderand/or beads, an alignment/imaging vision camera to view an image of thebead holder and/or beads, laser control on/off shutter, stage mechanicsto position the bead holder in the desired position for reading thebeads, and various optics to cause the excitation/read and imagingoptical signals to illuminate the beads and to allow the fluorescentoptical signals, imaging optical signals, and code related opticalsignals to be read by the appropriate devices, as described herein.

In addition, the microbead reader system 7 includes various electronics122 to provide any needed interfacing/buffering between the PC and theexternal devices and to perform the various functions described herein,including a junction box (optional) for interfacing between the computerand the optomechanical parts, an edge trigger circuit which receives thesignal from the edge trigger photodiode and provides a signal to thecomputer 104 indicative of when the incident light is incident on anaxial end edge of a bead, laser control electronics to control theon/off solenoid shutters 155 which control light from the green and redlasers, and photo-multiplier (PMT) control electronics to control thePMT's, e.g., to set the amount of gain on the PMTs.

Referring to FIG. 3, a block diagram of the opto-mechanical hardware 120(FIG. 2) is shown. In particular, there are two excitation lasers, agreen laser 152, e.g., a diode pumped frequency doubled Nd:YAG laserthat provides an output wavelength of about 532 nm (green) and has abeam waist of about 0.5 mm; and a red laser 150, e.g., a red Helium Neon(HeNe) laser that provides an output wavelength of about 633 nm (red)and has a beam waist of about 0.3 mm. Other beam sizes may be used ifdesired, provided it meets the performance/functions described hereinfor a given application. The output signals are processed through optics156,154, respectively, and passed to a polarization combiner 158 thatcombines both laser beams from the two lasers 150, 152 into a singlebeam. Alternatively, the combiner 158 may be a wavelength combiner;however in that case, the laser power cannot be adjusted by polarizationcontrol. The single beam is then provided to mode matching optics 160which creates a beam of the desired cross-sectional geometry (e.g.,elliptical) to illuminate the beads. The beam is also passed throughvarious routing mirrors 162 (discussed hereinafter) which routes thebeam to the desired location on the bead holder (or cuvette) 102. Thebead holder is positioned in the desired position to read a given bead,by the mechanical X-Y translation stage 112. The beads provide twooptical signals, the first is a diffracted code optical signal, similarto that discussed in the aforementioned patent applications, which ispassed to code pick-up optics 164 which routes the optical code signalto a code camera (or CCD camera) 168. The second optical signal providedfrom the beads is a fluorescence signal, which is passed to fluorescencepickup optics and passed along a multimode optical fiber 169, e.g., ThorM20L01, to PMT Receiver Module 170 which directs light from twodifferent wavelength fluorescent signals and provides each to a knownphotodetector, e.g., photomultiplier tubes (PMTs) discussed morehereinafter. Any photodetector having sufficient sensitivity may be usedif desired. The PMTs provide a signal to the computer indicative of thefluorescence signal from the beads 8. Also, the system may have analignment or imaging system 167 having an imaging camera for viewing thebeads in the cell 102 or for alignment or other purposes (discussedhereinafter).

Referring to FIG. 4, the laser block assembly comprises the lasers 150,152, optics 154 ,156 and polarization beam combiner 158, are shown. Inparticular, the green laser 152, e.g., a 532 nm laser LCM-T-11ccs, byPower Technology, provides a polarized optical laser signal to an on offShutter 201. When the shutter 201 is allowing light to pass, the light203 is passed to a ½ wave plate 200, e.g., CVI with a D=10 mm, which maybe rotated to adjust the power of the green laser 152. If the laserlight provided by one or both of the lasers 150, 152 is not polarized,optional polarizers 155 ,157 may be used to polarize the desired lightand then passed to the ½ wave plate. The wave plate 200 then providespolarization adjusted light to a focusing lens 202, e.g., f=150 mm PCX,D=25 mm Edmond Indust. Optics, which provides a converging or focussedbeam 203 to a 532 nm optical source filter then to a turning mirror 204.The distance between the green laser 152 and the wave plate 200 is about25 mm. The distance between the lens 202 to the doublet lens 218 isabout 115 mm.

The mirror 204 may be adjustable about one or more pivot points toensure that the beam 203 is incident on the correct location. The greenbeam 203 converges at a predetermined focal point F_(green) 220. Thedistance between the lens 202 and the polarizing cube 158 may beadjusted to place a focal point F_(green) 220 for the green beam 203 atthe desired focal location F_(green). The mirror 204 directs the beam203 onto the polarization combiner 158 (or cube).

The red laser 150, e.g., 633 mn JDSU 1.5 mWatt laser, provides apolarized optical laser light 213 to an on/off shutter 211. When theshutter 211 is allowing light to pass, the light 213 is passed to a 1/2wave plate 210 (same as the waveplate 200) which may be used to adjustthe power of the red laser 150. The wave plate 210 then providespolarization adjusted light to a focusing lens 212, e.g., f=75 mm PCXD=25 mm lens from Edmond Indust. Optics, which provides a converging orfocussed beam 213 to the polarization combiner 158 (or cube). The redbeam 213 converges at a predetermined focal point F_(red) 221 which isalso an adjustable focal point location set at or near to the samelocation as the focal point F_(green) 220 for the green beam 203. Thedistance between the lens 212 and the polarizing cube 158 may beadjusted to place a red laser focal point F_(red) 221 for the red beam213 at the desired location. The lens 212 is mounted to a Thor SPT1mount. The distance between the red laser 150 and the wave plate 210 isabout 25 mm. The distance between the lens 212 to the doublet lens 218is about 40 mm.

The shutters 201, 211 are controlled such that when the green laser isilluminating a given bead (for either code or fluorescence reading) thered laser is not also illuminating that bead at the same time. The twolasers 150, 152 may illuminate the same bead at the same time ifdesired, provided the fluorescent dyes used with the beads 8 arespectrally separated by a large enough wavelength space to allow theseparate dyes to be detected.

The polarization beam combiner 158 combines the two beams 203, 213 basedon their polarization and provides a combined beam 216, which isprovided to a doublet focusing lens 218, e.g., a 65 mm focal lengthdoublet lens, which works with the focusing lenses 202, 212 to focus thecombined beam 216 at a desired focal point 220 as a focused beam 219.The beam combiner 158 provides the light beam 216 as a circular beam andhas a distance of about 610 mm +/−10 mm to the bead 8 (not shown). Thepolarizing cube beam combiner 158 is mounted to a Thor Mount C4W.

Referring to FIG. 4 a, alternatively, the laser block assembly 159 maycomprise an alternative configuration as shown. In particular, the greenlaser 152 provides the light beams 304 to lens 300, e.g., a −50 mm F.L.lens Thor LD 1357, and then to a lens 302, e.g., a 50 mm FL lens Thor LB1844, and then to a flip mirror 310. When the flip mirror 310 is in theup position, the light 304 passes to a lens 312 and to a mirror 314 as abeam 306 to a lens 316 and to a turning mirror 318. The light 306 isreflected off the turning mirror 318 and provided to a lens 319 and to aprism 315, e.g., a 10 mm, 45 degree prism Edmond Ind. Optics NT32-325,which redirects the light 306 to a turning mirror 321 as the light beam306. The lenses 316, 319 are cylindrical lenses, e.g., Edmond Ind.Optics, NT46-017. The light beam 306 is used for reading the code in thebeads 8 as discussed herein and in the pending US patent applicationsreferenced herein. The lenses 300, 302 are used to accommodate orcompensate for beam tolerances in the green laser 152. When the flipmirror 310 is in the down position, the light 304 reflects off themirror 310 downward as a light 308 which is incident on a beam combiner,e.g., Chromatic (or wavelength) Beam Combiner Edmund Industries Optics,R47-265.

The red laser 150, e.g., a 635 nm Laser Sanyo DL-4148-21, provides a redlaser beam 324 to a lens 323, e.g., a 3.3 mm FL Lens Kodak A414TM. Thelight 324 then passes through lenses 320, 322, which may be the sametype as the lenses 300, 302, and are used to accommodate or compensatefor beam tolerances in the red laser 150. The light 324 is incident on acompensating glass optic 332, e.g., Edmond Ind. Optics, R47-265, whichremoves any astigmatism in the beam 324 that may be introduced by thechromatic beam combiner 334. The green light 308 and the red light 324are combined by the chromatic beam combiner 334 which provides acombined beam 326 to a lens 328, e.g., a 25 mm FL lens Thor AC127-025.The light 326 then passes to a turning mirror 330 and to a lens 336,e.g., a 75 mm FL lens Edmond Ind. Optics NT32-325. All the mirrors usedin FIG. 4 a are Edmond Ind. Optics R43-790. The beam combiner 334 isalso used to allow the red and green beams to share the same path, eventhough they may not both be traveling along that path at the same time.

The shutters 303, 323 are controlled such that when the green laser isilluminating a given bead (for either code or fluorescence reading) thered laser is not also illuminating that bead at the same time. The twolasers 150, 152 may illuminate the same bead at the same time ifdesired, provided the fluorescent dyes used with the beads 8 arespectrally separated by a large enough wavelength space to allow theseparate dyes to be detected.

Referring to FIG. 5, two side views of the combined beam 219 (from FIG.4) is shown as it would appear for light being incident on the end view(top portion of FIG. 5) and side view (bottom portion of FIG. 5) of abead 8. The combined beam 219 starting at the focal points F_(green)220, F_(red) 221, passes through a first cylindrical lens 222 and asecond cylindrical lens 224 which creates a focused beam 228 to aredirecting mirror 230 which is provided to the bead 8, having anelliptical bead spot geometry, with an end view 232 and a side view 234,designed to optimize the ability to read the bead code and thefluorescence with the same beam shape and minimal optical scatter. Suchbeam geometry is also discussed herein as well as in the aforementionedpatent application (U.S. patent application Ser. No. 10/661,235). Thecylindrical lenses 222,224 may be a f=150 mm cylindrical lens, 25 mmround; Edmunds E46-019. The focal point 221 lies along a virtual redpoint source plane 235 and the green focal point 220 lies along avirtual green point source plane 237. Also, the bead 8 is located at thefocal point of the beam 219, and lies in the image plane of the lenssystem. The distance from the virtual point source planes 235, 237 tothe first lens 222 is about 235 mm, the distance from the virtual pointsource planes 235,237 to the second lens 224 is about 415 mm, and thedistance from the virtual point source planes 235, 237 to the bead 8 isabout 650 mm. The two lenses 222, 224 allow the beam 228 size/geometryto be controlled independently in two different orthogonal optical axes.In addition, redirecting or routing or turning mirrors 234, 236 may beplaced between the cylindrical lenses 222 to provide the desired beampath for the desired mechanical layout for the reader system 7 (alsodiscussed hereinafter).

Referring to FIGS. 5A & 5B, a single beam shape or multiple differentbeam shapes may be used to read the code and fluorescence. Inparticular, in FIG. 5A, the beam 228 has a spot geometry 240 on a topview of the bead 8 as an elliptical shape, which is used for bothreading the code and reading the fluorescence of the bead 8. Wb=15microns for a 65 micron diameter bead (about 23% of the bead diameterD), and Lb=200 microns for a 450 microns long bead (about 40% of thebead length L). One problem with this approach is that, for fluorescencemeasurement, fluorescence from an adjacent bead may bleed or cross overto the current bead being read, thereby providing inaccurate beadfluorescence readings for the bead.

Referring to FIG. 5B, we have found that the beam spot size and shape onthe bead 8 may be optimized to provide improved fluorescence and codemeasurements by using a different shape beam for the code beam than thatused for the fluorescence beam. In particular, we have found that forreading the code, an elliptical beam shape 242 having a width Wb (1/e²full width of beam) that is about the same as the diameter D of the bead8 and a beam length Lb that is about 45% of the bead length L providesgood code read signals. The beam length Lb should not be so long as tocause the beam to scatter light off the edge of the bead being read intothe code reading optics/camera; and do not want the beam length Lb tooshort or the beam width Wb too narrow such that the bits cannot beresolved. The factors that affect this are as discussed in theaforementioned pending U.S. patent application Ser. No. 10/661,234,which is incorporated herein by reference in its entirety.

Regarding fluorescence, we have found having a beam width Wb about equalto the bead diameter D, provides the maximum amount of tolerance tovariations and inaccuracies between the beam and bead position forreading the fluorescence (i.e., transverse to the longitudinal axis ofthe bead), as discussed more hereinafter. Also, we have found that thebeam length Lb should be about less than about 14% of the bead length Lto minimize bead edge effects and thus optimize reading fluorescencealong the length of the bead 8, as discussed more hereinafter.Accordingly, the beams 244, 246 may be circular, or elliptical providedthe desired performance is obtained. For the red laser diode sourcediscussed herein the red beam is not circular and thus the beam at thebead is not circular; however this could be corrected optically ifdesired. The beam shapes for fluorescence reading is described morehereinafter.

Referring to FIG. 6, the combined excitation beam 228 is provided to therouting mirror 230, e.g., 1″ D×3 mmT ES 45-604, and directed to the bead8 which provides a transmitted beam 240 and a diffracted or reflectedbeam 242 from the bead code, as discussed in the aforementioned patentapplications. The reflected beam 242 is provided to a mirror 244, e.g,.1″ D×3 mmT ES 45-604, which provides the light to a bandpass filter 246,e.g., 532 nm BP filter ES NT47-136 (1″ Diam), which is adjacent to pairof lenses 247, 249, e.g., each a f-100 mm and each 25 mm diam ES 32-428.The bandpass filter 246 is designed to pass only the wavelength of lightassociated with the excitation/source set for reading the code. Thissubstantially eliminates the amount of optical noises/signal associatedwith other non-code reading wavelengths; thereby allowing a cleanoptical signal to pass to the code camera 168. The bandpass filter 246provides filtered light 248 to a beamsplitter 250, e.g., ES 43-817 25mm×1 mm R=25%, which reflects about 25% of the light along a path 252 tothe edge trigger photodiode 254, e.g., Sharp BS120 Digikey425-1001-5-ND. The diode 254 provides an electrical signal on a line 255to the computer indicative of the intensity of the light. The remainderof the input light 248 passes straight through the beamsplitter 250 as abeam 256 which creates a bead code image at a predetermined focal point258. The lens pair around the bandpass filter 246 transfers the image ofthe reflected beam at the bead 8 to the bead code image point 258 aswell as on the edge pick-up diode 254. The light 256 is provided to avideo lens 260, e.g., Computer V1213 f=12.5, which provides a focusedoptical signal on the code camera 168, e.g., Lumera LU-050M. The videolens 260 is used as a Fourier lens to project the Fourier transform ofthe bead code from the point 258 onto the code camera 262. The codecamera 262 provides a digital signal on a line 264 to the computerindicative of the bead code image at the point 258.

Referring to FIG. 6A, the BP filter 246 can be anywhere in the code pathas indicated by the numerals 261, 263, 265, 267, provided it does notsignificantly deteriorate wavefront performance of the optical system ordegrade the lens performance. The two f=100 mm lenses 247, 249 are fortransferring the image from the bead 8 to the intermediate point 258.Thus, the distance from the lens 249 to the virtual image point 258 is100 mm and the distance from the bead 8 to the lens 247 is about 100 mm(equal to the focal length of the lenses 247, 249). Also, the focallengths of the two lenses 247 ,249 need not be the same, providedappropriate distance compensation is performed, and also depending onthe application and performance specifications. Also, the distance fromthe point 258 to the video lens 260 is about 12.5 mm and the distancefrom the video lens to the image plane on the camera (and the FourierPlane) 269, is about 12.5 mm (equal to the focal length of the videolens 260). Technically, the lenses 247 ,249 should be separated by 2*fin order to yield a Fourier Transform at the image plane 269 of the codecamera 168 (a typical 4f system). However, this configuration does notcause the beam waist to change substantially, thereby not significantlyaltering the performance of the Fourier transform. It should beunderstood that in FIG. 6A, the light travels from left to right, withconnects with the prior optical drawing of FIG. 5. However, in theactual hardware shown in the hardware FIG. 6, the bead 8 would be on theright side and the light would travel from right to left.

Referring to FIG. 6, the excitation beam 228 also excites fluorescentmolecules attached to the bead 8, which provide a fluorescent opticalsignal 268 to a fluorescence pick-up head 166, having a collimator,which directs the fluorescent optical signal into an optical fiber,e.g., a multimode optical fiber, which is provided to PMT optics,discussed hereinafter.

More specifically, referring to FIG. 6B, the fluorescent signal isprovided to a collection objective lens 280, e.g., Lightpath (Geltech)350220, F=11 mm asphere NA=0.25, which provides light to a longwavelength pass filter 282, e.g,. 0.5″ diam. filter glass made by SchottPart No. OG-570, to prevent excitation light at 532 nm from getting intothe fiber and causing the cladding to fluoresce. If the fiber is made ofall glass, the filter is likely not needed. The collection angle θc forlight to enter the fiber is set to about 30 degrees based on apredetermined numerical aperture (NA). Other values for the collectionangle θc may be used depending on the amount of stray light and therequired detection performance. The light then passes to a fiberfocusing assembly 284, e.g., Thor M15L01, which focuses the fluorescentlight 268 into the end of the fiber 169. The collimator assembly 166that may be used is a Thor F220-SMA-A Collimator.

In addition, when it is desired to view a visible image of the beads inthe bead holder (e.g., for alignment, bead counting, or other purposes),a white LED 270, e.g,. Lumex SSL-LX5093XUWC, is illuminated whichprovides a white light illumination signal 272 up through the bottom ofthe bead holder and beads to illuminate the beads 8. The LED 270 ismounted to a PMT shutter (discussed hereinafter) which allows it to flipout of the way when fluorescence is being detected. The illuminationimage signal 272 is provided to a mirror 274 which reflects the light272 through a first lens 279, e.g., Infinity 0.75× lens, and a secondlens, e.g., Infinity 2× lens, and then onto an imaging/vision camera276, e.g., Lumera LU-050C. The vision camera 276 provides an electricalsignal on a line 278 to the computer indicative of the image seen by theimaging camera 276.

It should be understood that the alignment camera 276 may be on the sameside of the bead 8 (or bead holder 102) as the fluorescent pick-up 269.Alternatively, the location of the alignment camera 276 and fluorescentpick-up 269 may be swapped, such that the alignment camera 276 isbeneath the bead 8 and the pick-up 269 is above the bead 8. It should beunderstood that one can swap the incident beam 228 and the reflectedbeam 242 and the associated optics.

When the bead 8 is not present, the transmitted beam 240 may be incidenton a laser power diode 243, e.g., Hamamatsu S2307-16R, which provides anelectrical signal on a line 241 proportional to the power of theincident beam 228. This may be used for laser power calibration or othersystem calibration or test purposes. This light beam 228 may also beused for edge trigger information, as discussed hereafter.

Referring to FIG. 7, the light from the fluorescent pick-up head 166 isprovided along the fiber 169 e.g., Thor M20L01 multimode fiber, to a PMTreceiver module 170, which includes a collimator assembly 281, e.g.,Thor F230SMA-A, and a focusing lens 282, e.g., f-100 mm, 25 mm diam. ES32-482, which provides light 485 to a dichroic beam splitter 284, e.g.,Omega 630 DLRP XF2021, 21×29 mm, 1 mm thick. The distance from thecollimator assembly 281 to the beam splitter 284 is about 1 to 2 inches.The beam splitter 284 reflects light 286 of a first wavelength (e.g.,green pumped Cy3 fluorescent light), and passes light 288 of a secondwavelength (e.g., red pumped Cy5 fluorescent light). The light 286 ispassed through an optical aperture, e.g., 12.5 mm Aperture Thor SM1A5,and then through an optical filter, e.g., Omega 3RD-570LP-610SP, 25 mmdiam, about 3 mm thick, that passes light of the first wavelength (e.g.,green pumped Cy3 fluorescent light), to a photomultiplier tube (PMT)292, e.g., Hamamatsu H5783-20. The PMT 292 detects the intensity of theincident fluorescent light and provides an output electrical signal on aline 293 to the computer indicative of the intensity of the fluorescencesignal incident on the PMT 292.

Similarly, the light 288 passes through an optical aperture, e.g., 12.5mm Aperture Thor SM1A5, and then through a filter glass RG645, 1″ diam,1 mm thick and then through an optical filter, e.g., Omega 695AF55,XF3076, 25 mm diam, about 3 mm thick, that passes light of the secondwavelength (e.g., red pumped CyS fluorescent light), to a secondphotomultiplier tube (PMT) 296, e.g., Hamamatsu H5783-20. The PMT 296detects the intensity of the incident fluorescent light and provides anoutput electrical signal on a line 293 to the computer indicative of theintensity of the fluorescence signal incident on the PMT 296.

It should be understood that fluorescent molecules that are excited bythe 532 nm (green) laser produce a fluorescent signal having awavelength of about 570 nm (orange color), and fluorescent moleculesthat are excited by the 633 nm (red) laser produce a fluorescent signalhaving a wavelength of about 670 nm (deep red color). Accordingly, thefluorescent signal on the line 286 will have an orange color and thelight 288 will be deep red.

Referring to FIGS. 8-22, show various perspective and cutaway views ofthe present invention. It also shows the path of the light beams fromvarious views.

Referring to FIGS. 8-10, perspective views of one embodiment of thepresent invention, which shows numerous parts having the same numeralsas in other Figs. herein, and also shows, a green laser power supply andcontrol 402, red laser power supply and control 406, a frame or housing410, and a main circuit board 410. FIG. 12 is a physical drawing of oneembodiment of FIG. 4 laser block assembly.

The parts used for the present invention are known parts and may besubstituted for other parts that provide the same function as thatdescribed herein, unless stated otherwise.

For example, as discussed herein, the code camera may be a U.S.B 2.0camera, comprising a Luminera Monochromatic camera; part no. LU-050M,coupled to a Computar 12.5 mm focal length TV lens. The camera providesa U.S.B 2.0 (universal serial bus) serial data stream indicative of theimage seen by the camera. Alternatively, the camera may be a standardCCD camara, or a CCD linear array, part No. CCD 111 made by FairchildImaging Corp., or other camera capable of providing a digital or analogsignal indicative of the image seen, having sufficient resolution toidentify the bits in the code in the beads 8. In that case, a “framegrabber” and A/D converter may be needed within the computer to properlycondition the code signal for processing. In addition, the cameraaccepts a trigger signal to command the camera to capture or save ortransmit the image seen by the camera. The image or vision camera may bea Luminera LU-050C, U.S.B 2.0 color camera. The X-Y translation stagemay be a Ludl X-Y precision stage driver/controller, having motordrives, position feedback and limit signals. Any other x-y stage may beused if desired, provided the stage can be positioned with sufficientaccuracy to accurately read the beads 8.

The adjustable focus lenses described herein allow the setting of thespot size and focal point for the green and red laser light. Oneembodiment of the system described herein has three shutters that arecontrolled by the computer, one for each laser and one to prevent lightfrom getting to the PMTs. This shutter also holds the white light sourcediscussed herein for the bead Imaging System.

Referring to FIG. 23, a diffraction grating-based optical identificationelement 8 (or encoded element or coded element) comprises a knownoptical substrate 10, having an optical diffraction grating 12 disposed(or written, impressed, embedded, imprinted, etched, grown, deposited orotherwise formed) in the volume of or on a surface of a substrate 10.The grating 12 is a periodic or a periodic variation in the effectiverefractive index and/or effective optical absorption of at least aportion of the substrate 10.

The optical identification element described herein is the same as thatdescribed in Copending patent application Ser. No., filedcontemporaneously herewith, which is incorporated herein by reference inits entirety.

In particular, the substrate 10 has an inner region 20 where the grating12 is located. The inner region 20 may be photosensitive to allow thewriting or impressing of the grating 12. The substrate 10 has an outerregion 18 which does not have the grating 12 therein.

The grating 12 is a combination of one or more individual spatialperiodic sinusoidal variations (or components) in the refractive indexthat are collocated at substantially the same location on the substrate10 along the length of the grating region 20, each having a spatialperiod (or pitch) Λ. The resultant combination of these individualpitches is the grating 12, comprising spatial periods (Λ1-Λn) eachrepresenting a bit in the code. Thus, the grating 12 represents a uniqueoptically readable code, made up of bits, where a bit corresponds to aunique pitch Λ within the grating 12. Accordingly, for a digital binary(0-1) code, the code is determined by which spatial periods (Λ1-Λn)exist (or do not exist) in a given composite grating 12. The code orbits may also be determined by additional parameters (or additionaldegrees of multiplexing), and other numerical bases for the code may beused, as discussed herein and/or in the aforementioned patentapplication.

The grating 12 may also be referred to herein as a composite orcollocated grating. Also, the grating 12 may be referred to as a“hologram”, as the grating 12 transforms, translates, or filters aninput optical signal to a predetermined desired optical output patternor signal.

The substrate 10 has an outer diameter D1 and comprises silica glass(SiO₂) having the appropriate chemical composition to allow the grating12 to be disposed therein or thereon. Other materials for the opticalsubstrate 10 may be used if desired. For example, the substrate 10 maybe made of any glass, e.g., silica, phosphate glass, borosilicate glass,or other glasses, or made of glass and a polymer, or solely a polymer.For high temperature or harsh chemical applications, the opticalsubstrate 10 made of a glass material is desirable. If a flexiblesubstrate is needed, plastic, rubber or polymer-based substrate may beused. The optical substrate 10 may be any material capable of having thegrating 12 disposed in the grating region 20 and that allows light topass through it to allow the code to be optically read.

The optical substrate 10 with the grating 12 has a length L and an outerdiameter D1, and the inner region 20 diameter D. The length L can rangefrom very small “microbeads” (or microelements, micro-particles, orencoded particles), about 1-1000 microns or smaller, to larger“macroelements” for larger applications (about 1.0-1000 mm or greater).In addition, the outer dimension D1 can range from small (less than 1000microns) to large (1.0-1000 mm and greater). Other dimensions andlengths for the substrate 10 and the grating 12 may be used.

The optical substrate 10 with the grating 12 has a length L and an outerdiameter D1, and the inner region 20 diameter D. The length L can rangefrom very small (about 1-1000 microns or smaller) to large (about1.0-1000 mm or greater). In addition, the outer dimension D1 can rangefrom small (less than 1000 microns) to large (1.0-1000 mm and greater).Other dimensions and lengths for the substrate 10 and the grating 12 maybe used. Also, the element may be embedded within or part of a largersubstrate or object. The element may also be in the form of a thread orfiber to be weaved into a material.

Some non-limiting examples of microbeads discussed herein are about 28microns diameter and about 250 microns long, and about 65 micronsdiameter and about 400 microns long. Other lengths may be used asdiscussed herein.

The grating 12 may have a length Lg of about the length L of thesubstrate 10. Alternatively, the length Lg of the grating 12 may beshorter than the total length L of the substrate 10.

The outer region 18 is made of pure silica (SiO₂) and has a refractiveindex n2 of about 1.458 (at a wavelength of about 1553 nm), and theinner grating region 20 of the substrate 10 has dopants, such asgermanium and/or boron, to provide a refractive index n1 of about 1.453,which is less than that of outer region 18 by about 0.005. Other indicesof refraction n1,n2 for the grating region 20 and the outer region 18,respectively, may be used, if desired, provided the grating 12 can beimpressed in the desired grating region 20. For example, the gratingregion 20 may have an index of refraction that is larger than that ofthe outer region 18 or grating region 20 may have the same index ofrefraction as the outer region 18 if desired.

Referring to FIG. 24, an incident light 24 of a wavelength λ, e.g., 532nm from a known frequency doubled Nd:YAG laser or 632 nm from a knownHelium-Neon laser, is incident on the grating 12 in the substrate 10.Any other input wavelength λ can be used if desired provided λ is withinthe optical transmission range of the substrate (discussed more hereinand/or in the aforementioned patent application). A portion of the inputlight 24 passes straight through the grating 12, as indicated by a line25. The remainder of the input light 24 is reflected by the grating 12,as indicated by a line 27 and provided to a detector 29. The outputlight 27 may be a plurality of beams, each having the same wavelength λas the input wavelength λ and each having a different output angleindicative of the pitches (Λ1-Λn) existing in the grating 12.Alternatively, the input light 24 may be a plurality of wavelengths andthe output light 27 may have a plurality of wavelengths indicative ofthe pitches (Λ1-Λn) existing in the grating 12. Alternatively, theoutput light may be a combination of wavelengths and output angles. Theabove techniques are discussed in more detail herein and/or in theaforementioned patent application.

The detector 29 has the necessary optics, electronics, software and/orfirmware to perform the functions described herein. In particular, thedetector reads the optical signal 27 diffracted or reflected from thegrating 12 and determines the code based on the pitches present or theoptical pattern, as discussed more herein or in the aforementionedpatent application. An output signal indicative of the code is providedon a line 31.

Referring to FIG. 25, The reflected light 27, comprises a plurality ofbeams 26-36 that pass through a lens 37, which provides focused lightbeams 46-56, respectively, which are imaged onto a CCD camera 60. Thelens 37 and the camera 60, and any other necessary electronics or opticsfor performing the functions described herein, make up the reader 29.Instead of or in addition to the lens 37, other imaging optics may beused to provide the desired characteristics of the optical image/signalonto the camera 60 (e.g., spots, lines, circles, ovals, etc.), dependingon the shape of the substrate 10 and input optical signals. Also,instead of a CCD camera other devices may be used to read/capture theoutput light.

Referring to FIG. 26, the image on the CCD camera 60 is a series ofilluminated stripes indicating ones and zeros of a digital pattern orcode of the grating 12 in the element 8. Referring to FIG. 27, lines 68on a graph 70 are indicative of a digitized version of the image of FIG.26 as indicated in spatial periods (Λ1 -Λn).

Each of the individual spatial periods (Λ1-Λn) in the grating 12 isslightly different, thus producing an array of N unique diffractionconditions (or diffraction angles) discussed more hereinafter. When theelement 8 is illuminated from the side, in the region of the grating 12,at an appropriate input angle, e.g., about 30 degrees, with a singleinput wavelength λ (monochromatic) source, the diffracted (or reflected)beams 26-36 are generated. Other input angles θi may be used if desired,depending on various design parameters as discussed herein and/or in theaforementioned patent application, and provided that a known diffractionequation (Eq. 1 below) is satisfied:sin(θ_(i))+sin(θ_(o))=mλ/nΛ  Eq. 1where Eq. 1 is diffraction (or reflection or scatter) relationshipbetween input wavelength λ, input incident angle θi, output incidentangle θo, and the spatial period Λ of the grating 12. Further, m is the“order” of the reflection being observed, and n is the refractive indexof the substrate 10. The value of m=1 or first order reflection isacceptable for illustrative purposes. Eq. 1 applies to light incident onouter surfaces of the substrate 10 which are parallel to thelongitudinal axis of the grating (or the k_(B) vector). Because theangles θi,θo are defined outside the substrate 10 and because theeffective refractive index of the substrate 10 is substantially a commonvalue, the value of n in Eq. 1 cancels out of this equation.

Thus, for a given input wavelength λ, grating spacing Λ, and incidentangle of the input light θi, the angle θo of the reflected output lightmay be determined. Solving Eq. 1 for θo and plugging in m=1, gives:θo=sin⁻¹(λ/Λ−sin(θi))  Eq. 2For example, for an input wavelength λ=532 nm, a grating spacing Λ=0.532microns (or 532 nm), and an input angle of incidence θi=30 degrees, theoutput angle of reflection will be θo=30 degrees. Alternatively, for aninput wavelength λ=632 nm, a grating spacing Λ=0.532 microns (or 532nm), and an input angle θi of 30 degrees, the output angle of reflectionθo will be at 43.47 degrees, or for an input angle θi=37 degrees, theoutput angle of reflection will be θo=37 degrees. Any input angle thatsatisfies the design requirements discussed herein and/or in theaforementioned patent application may be used.

In addition, to have sufficient optical output power and signal to noiseratio, the output light 27 should fall within an acceptable portion ofthe Bragg envelope (or normalized reflection efficiency envelope) curve200, as indicated by points 204, 206, also defined as a Bragg envelopeangle θB, as also discussed herein and/or in the aforementioned patentapplication. The curve 200 may be defined as:

$\begin{matrix}{{I( {{ki},{ko}} )} \approx {\lbrack{KD}\rbrack^{2}\mspace{11mu}\sin\;{c^{2}\lbrack \frac{( {{ki} - {ko}} )\; D}{2} \rbrack}}} & {{Eq}.\mspace{14mu} 3}\end{matrix}$where K=2πδn/λ, where, δn is the local refractive index modulationamplitude of the grating and λ is the input wavelength,sinc(x)=sin(x)/x, and the vectors k_(i)=2π cos(θ_(i))/λ and k_(o)=2π cos(θ_(o))/λ are the projections of the incident light and the output (orreflected) light, respectively, onto the line 203 normal to the axialdirection of the grating 12 (or the grating vector k_(B)), D is thethickness or depth of the grating 12 as measured along the line 203(normal to the axial direction of the grating 12). Other substrateshapes than a cylinder may be used and will exhibit a similar peakedcharacteristic of the Bragg envelope. We have found that a value for δnof about 10⁻⁴ in the grating region of the substrate is acceptable;however, other values may be used if desired.

Rewriting Eq. 3 gives the reflection efficiency profile of the Braggenvelope as:

$\begin{matrix}{{I( {{ki},{ko}} )} \approx {\lbrack \frac{2\;{\pi \cdot \delta}\;{n \cdot D}}{\lambda} \rbrack^{2}\lbrack \frac{{Sin}(x)}{x} \rbrack}^{2}} & {{Eq}.\mspace{14mu} 4}\end{matrix}$where:x=(ki−ko)D/2=(πD/λ)*(cos θi−cos θo)

Thus, when the input angle θi is equal to the output (or reflected)angle θ_(o) (i.e., θi=θ_(o), the reflection efficiency I (Eqs. 3 & 4) ismaximized, which is at the center or peak of the Bragg envelope. Whenθi=θo, the input light angle is referred to as the Bragg angle as isknown. The efficiency decreases for other input and output angles (i.e.,θi≠θ_(o)), as defined by Eqs. 3 & 4. Thus, for maximum reflectionefficiency and thus output light power, for a given grating pitch Λ andinput wavelength, the angle θi of the input light 24 should be set sothat the angle θo of the reflected output light equals the input angleθi.

Also, as the thickness or diameter D of the grating decreases, the widthof the sin(x)/x function (and thus the width of the Bragg envelope)increases and, the coefficient to or amplitude of the sinc² (or(sin(x)/x)² f2unction (and thus the efficiency level across the Braggenvelope) also increases, and vice versa. Further, as the wavelength λincreases, the half-width of the Bragg envelope as well as theefficiency level across the Bragg envelope both decrease. Thus, there isa trade-off between the brightness of an individual bit and the numberof bits available under the Bragg envelope. Ideally, δn should be madeas large as possible to maximize the brightness, which allows D to bemade smaller.

From Eq. 3 and 4, the half-angle of the Bragg envelope θ_(B) is definedas:

$\begin{matrix}{\theta_{B} = \frac{\eta\;\lambda}{\pi\; D\mspace{11mu}\sin\;( \theta_{i} )}} & {{Eq}.\mspace{14mu} 5}\end{matrix}$

where η is a reflection efficiency factor which is the value for x inthe sinc²(x) function where the value of sinc²(x) has decreased to apredetermined value from the maximum amplitude as indicated by points204, 206 on the curve 200.

We have found that the reflection efficiency is acceptable when η≦1.39.This value for η corresponds to when the amplitude of the reflected beam(i.e., from the sinc²(x) function of Eqs. 3 & 4) has decayed to about50% of its peak value. In particular, when x=1.39=η, sinc²(x)=0.5.However, other values for efficiency thresholds or factor in the Braggenvelope may be used if desired.

The beams 26-36 are imaged onto the CCD camera 60 to produce the patternof light and dark regions 120-132 representing a digital (or binary)code, where light=1 and dark=0 (or vice versa). The digital code may begenerated by selectively creating individual index variations (orindividual gratings) with the desired spatial periods Λ1-Λn. Otherillumination, readout techniques, types of gratings, geometries,materials, etc. may be used as discussed in the aforementioned patentapplication.

Referring to FIG. 28, illustrations (a)-(c), for the grating 12 in acylindrical substrate 10 having a sample spectral 17 bit code (i.e., 17different pitches Λ1-Λ17), the corresponding image on the CCD (ChargeCoupled Device) camera 60 is shown for a digital pattern of 7 bitsturned on (10110010001001001); 9 bits turned on of (1100010101010011);all 17 bits turned on of (11111111111111111).

For the images in FIG. 28, the length of the substrate 10 was 450microns, the outer diameter D1 was 65 microns, the inner diameter D was14 microns, δn for the grating 12 was about 10⁻⁴, n1 in portion 20 wasabout 1.458 (at a wavelength of about 1550 nm), n2 in portion 18 wasabout 1.453, the average pitch spacing Λ for the grating 12 was about0.542 microns, and the spacing between pitches ΔΛ was about 0.36% of theadjacent pitches Λ.

Referring to FIG. 29, illustration (a), the pitch Λ of an individualgrating is the axial spatial period of the sinusoidal variation in therefractive index n1 in the region 20 of the substrate 10 along the axiallength of the grating 12 as indicated by a curve 90 on a graph 91.Referring to FIG. 29, illustration (b), a sample composite grating 12comprises three individual gratings that are co-located on the substrate10, each individual grating having slightly different pitches, Λ1, Λ2,Λ3, respectively, and the difference (or spacing) ΔΛ between each pitchΛ being about 3.0% of the period of an adjacent pitch Λ as indicated bya series of curves 92 on a graph 94. Referring to FIG. 29, illustration(c), three individual gratings, each having slightly different pitches,Λ1, Λ2, Λ3, respectively, are shown, the difference ΔΛ between eachpitch Λ being about 0.3% of the pitch Λ of the adjacent pitch as shownby a series of curves 95 on a graph 97. The individual gratings in FIG.29, illustrations (b) and (c) are shown to all start at 0 forillustration purposes; however, it should be understood that, theseparate gratings need not all start in phase with each other. Referringto FIG. 29, illustration (d), the overlapping of the individualsinusoidal refractive index variation pitches Λ1-Λn in the gratingregion 20 of the substrate 10, produces a combined resultant refractiveindex variation in the composite grating 12 shown as a curve 96 on agraph 98 representing the combination of the three pitches shown in FIG.29, illustration (b). Accordingly, the resultant refractive indexvariation in the grating region 20 of the substrate 10 may not besinusoidal and is a combination of the individual pitches Λ (or indexvariation).

The maximum number of resolvable bits N, which is equal to the number ofdifferent grating pitches Λ (and hence the number of codes), that can beaccurately read (or resolved) using side-illumination and side-readingof the grating 12 in the substrate 10, is determined by numerousfactors, including: the beam width w incident on the substrate (and thecorresponding substrate length L and grating length Lg), the thicknessor diameter D of the grating 12, the wavelength λ of incident light, thebeam divergence angle θ_(R), and the width of the Bragg envelope θ_(B)(discussed more in the aforementioned patent application), and may bedetermined by the equation:

$\begin{matrix}{N \cong \frac{\eta\;\beta\; L}{2\; D\mspace{11mu}{\sin( \theta_{i} )}}} & {{Eq}.\mspace{14mu} 6}\end{matrix}$

Referring to 30, illustration (b), the transmission wavelength spectrumof the transmitted output beam 330 (which is transmitted straightthrough the grating 12) will exhibit a series of notches (or dark spots)696. Alternatively, instead of detecting the reflected output light 310,the transmitted light 330 may be detected at the detector/reader 308. Itshould be understood that the optical signal levels for the reflectionpeaks 695 and transmission notches 696 will depend on the “strength” ofthe grating 12, i.e., the magnitude of the index variation n in thegrating 12.

Referring to FIG. 31, instead of or in addition to the pitches Λ in thegrating 12 being oriented normal to the longitudinal axis, the pitchesmay be created at a angle θg. In that case, when the input light 24 isincident normal to the surface 792, will produce a reflected output beam790 having an angle θo determined by Eq. 1 as adjusted for the blazeangle θg. This can provide another level of multiplexing bits in thecode.

The grating 12 may be impressed in the substrate 10 by any technique forwriting, impressed, embedded, imprinted, or otherwise forming adiffraction grating in the volume of or on a surface of a substrate 10.Examples of some known techniques are described in U.S. Pat. Nos.4,725,110 and 4,807,950, entitled “Method for Impressing Gratings WithinFiber Optics”, to Glenn et al; and U.S. Pat. No. 5,388,173, entitled“Method and Apparatus for Forming A periodic Gratings in OpticalFibers”, to Glenn, respectively, and U.S. Pat. No. 5,367,588, entitled“Method of Fabricating Bragg Gratings Using a Silica Glass Phase GratingMask and Mask Used by Same”, to Hill, and U.S. Pat. No. 3,916,182,entitled “Periodic Dielectric Waveguide Filter”, Dabby et al, and U.S.Pat. No. 3,891,302, entitled “Method of Filtering Modes in OpticalWaveguides”, to Dabby et al, which are all incorporated herein byreference to the extent necessary to understand the present invention.

Alternatively, instead of the grating 12 being impressed within thesubstrate material, the grating 12 may be partially or totally createdby etching or otherwise altering the outer surface geometry of thesubstrate to create a corrugated or varying surface geometry of thesubstrate, such as is described in U.S. Pat. No. 3,891,302, entitled“Method of Filtering Modes in Optical Waveguides”, to Dabby et al, whichis incorporated herein by reference to the extent necessary tounderstand the present invention, provided the resultant opticalrefractive profile for the desired code is created.

Further, alternatively, the grating 12 may be made by depositingdielectric layers onto the substrate, similar to the way a known thinfilm filter is created, so as to create the desired resultant opticalrefractive profile for the desired code.

The substrate 10 (and/or the element 8) may have end-viewcross-sectional shapes other than circular, such as square, rectangular,elliptical, clam-shell, D-shaped, or other shapes, and may haveside-view sectional shapes other than rectangular, such as circular,square, elliptical, clam-shell, D-shaped, or other shapes. Also, 3Dgeometries other than a cylinder may be used, such as a sphere, a cube,a pyramid or any other 3D shape. Alternatively, the substrate 10 mayhave a geometry that is a combination of one or more of the foregoingshapes.

The shape of the element 8 and the size of the incident beam may be madeto minimize any end scatter off the end face(s) of the element 8, as isdiscussed herein and/or in the aforementioned patent application.Accordingly, to minimize such scatter, the incident beam 24 may be ovalshaped where the narrow portion of the oval is smaller than the diameterD1, and the long portion of the oval is smaller than the length L of theelement 8. Alternatively, the shape of the end faces may be rounded orother shapes or may be coated with an antireflective coating.

It should be understood that the size of any given dimension for theregion 20 of the grating 12 may be less than any corresponding dimensionof the substrate 10. For example, if the grating 12 has dimensions oflength Lg, depth Dg, and width Wg, and the substrate 12 has differentdimensions of length L, depth D, and width W, the dimensions of thegrating 12 may be less than that of the substrate 12. Thus, the grating12, may be embedded within or part of a much larger substrate 12. Also,the element 8 may be embedded or formed in or on a larger object foridentification of the object.

The dimensions, geometries, materials, and material properties of thesubstrate 10 are selected such that the desired optical and materialproperties are met for a given application. The resolution and range forthe optical codes are scalable by controlling these parameters asdiscussed herein and/or in the aforementioned patent application.

Referring to FIG. 32, the substrate 10 may have an outer coating 799,such as a polymer or other material that may be dissimilar to thematerial of the substrate 10, provided that the coating 799 on at leasta portion of the substrate, allows sufficient light to pass through thesubstrate for adequate optical detection of the code. The coating 799may be on any one or more sides of the substrate 10. Also, the coating799 may be a material that causes the element 8 to float or sink incertain fluids (liquid and/or gas) solutions.

Also, the substrate 10 may be made of a material that is less dense thancertain fluid (liquids and/or gas) solutions, thereby allowing theelements 8 to float or be buoyant or partially buoyant. Also, thesubstrate may be made of a porous material, such as controlled poreglass (CPG) or other porous material, which may also reduce the densityof the element 8 and may make the element 8 buoyant or partially-buoyantin certain fluids.

Also, the grating 12 is axially spatially invariant. As a result, thesubstrate 10 with the grating 12 may be axially subdivided or cut intomany separate smaller substrates and each substrate will contain thesame code as the longer substrate had before it was cut. The limit onthe size of the smaller substrates is based on design and performancefactors discussed herein and/or in the aforementioned patentapplication.

Referring to FIG. 33, one purpose of the outer region 18 (or regionwithout the grating 12) of the substrate 10 is to provide mechanical orstructural support for the inner grating region 20. Accordingly, theentire substrate 10 may comprise the grating 12, if desired.Alternatively, the support portion may be completely or partiallybeneath, above, or along one or more sides of the grating region 20,such as in a planar geometry, or a D-shaped geometry, or othergeometries, as described herein and/or in the aforementioned patentapplication. The non-grating portion 18 of the substrate 10 may be usedfor other purposes as well, such as optical lensing effects or othereffects (discussed herein or in the aforementioned patent application).Also, the end faces of the substrate 10 need not be perpendicular to thesides or parallel to each other. However, for applications where theelements 8 are stacked end-to-end, the packing density may be optimizedif the end faces are perpendicular to the sides.

Referring to FIG. 34, illustrations (a)-(c), two or more substrates 10,250, each having at least one grating therein, may be attached togetherto form the element 8, e.g., by an adhesive, fusing or other attachmenttechniques. In that case, the gratings 12, 252 may have the same ordifferent codes.

Referring to FIGS. 35, illustrations (a) and (b), the substrate 10 mayhave multiple regions 80, 90 and one or more of these regions may havegratings in them. For example, there may be gratings 12, 252side-by-side (illustration (a)), or there may be gratings 82-88, spacedend-to-end (illustration (b)) in the substrate 10.

Referring to FIG. 36, the length L of the element 8 may be shorter thanits diameter D, thus, having a geometry such as a plug, puck, wafer,disc or plate.

Referring to FIG. 37, to facilitate proper alignment of the grating axiswith the angle θi of the input beam 24, the substrate 10 may have aplurality of the gratings 12 having the same codes written therein atnumerous different angular or rotational (or azimuthal) positions of thesubstrate 10. In particular, two gratings 550, 552, having axial gratingaxes 551, 553, respectively may have a common central (or pivot orrotational) point where the two axes 551, 553 intersect. The angle θi ofthe incident light 24 is aligned properly with the grating 550 and isnot aligned with the grating 552, such that output light 555 isreflected off the grating 550 and light 557 passes through the grating550 as discussed herein. If the element 8 is rotated as shown by thearrows 559, the angle θi of incident light 24 will become alignedproperly with the grating 552 and not aligned with the grating 550 suchthat output light 555 is reflected off the grating 552 and light 557passes through the grating 552. When multiple gratings are located inthis rotational orientation, the bead may be rotated as indicated by aline 559 and there may be many angular positions that will providecorrect (or optimal) incident input angles θi to the grating. While thisexample shows a circular cross-section, this technique may be used withany shape cross-section.

Referring to FIG. 38, illustrations (a), (b), (c), (d), and (e) thesubstrate 10 may have one or more holes located within the substrate 10.In illustration (a), holes 560 may be located at various points alongall or a portion of the length of the substrate 10. The holes need notpass all the way through the substrate 10. Any number, size and spacingfor the holes 560 may be used if desired. In illustration (b), holes 572may be located very close together to form a honeycomb-like area of allor a portion of the cross-section. In illustration (c), one (or more)inner hole 566 may be located in the center of the substrate 10 oranywhere inside of where the grating region(s) 20 are located. The innerhole 566 may be coated with a reflective coating 573 to reflect light tofacilitate reading of one or more of the gratings 12 and/or to reflectlight diffracted off one or more of the gratings 12. The incident light24 may reflect off the grating 12 in the region 20 and then reflect offthe surface 573 to provide output light 577. Alternatively, the incidentlight 24 may reflect off the surface 573, then reflect off the grating12 and provide the output light 575. In that case the grating region 20may run axially or circumferentially 571 around the substrate 10. Inillustration (d), the holes 579 may be located circumferentially aroundthe grating region 20 or transversely across the substrate 10. Inillustration (e), the grating 12 may be located circumferentially aroundthe outside of the substrate 10, and there may be holes 574 inside thesubstrate 10.

Referring to FIG. 39, illustrations (a), (b), and (c), the substrate 10may have one or more protruding portions or teeth 570, 578, 580extending radially and/or circumferentially from the substrate 10.Alternatively, the teeth 570, 578, 580 may have any other desired shape.

Referring to FIG. 40, illustrations (a), (b), (c) a D-shaped substrate,a flat-sided substrate and an eye-shaped (or clam-shell or teardropshaped) substrate 10, respectively, are shown. Also, the grating region20 may have end cross-sectional shapes other than circular and may haveside cross-sectional shapes other than rectangular, such as any of thegeometries described herein for the substrate 10. For example, thegrating region 20 may have a oval cross-sectional shape as shown bydashed lines 581, which may be oriented in a desired direction,consistent with the teachings herein. Any other geometries for thesubstrate 10 or the grating region 20 may be used if desired, asdescribed herein.

Referring to FIG. 41, at least a portion of a side of the substrate 10may be coated with a reflective coating to allow incident light 510 tobe reflected back to the same side from which the incident light came,as indicated by reflected light 512.

Referring to FIG. 42, illustrations (a) and (b), alternatively, thesubstrate 10 can be electrically and/or magnetically polarized, by adopant or coating, which may be used to ease handling and/or alignmentor orientation of the substrate 10 and/or the grating 12, or used forother purposes. Alternatively, the bead may be coated with conductivematerial, e.g., metal coating on the inside of a holy substrate, ormetallic dopant inside the substrate. In these cases, such materials cancause the substrate 10 to align in an electric or magnetic field.Alternatively, the substrate can be doped with an element or compoundthat fluoresces or glows under appropriate illumination, e.g., a rareearth dopant, such as Erbium, or other rare earth dopant or fluorescentor luminescent molecule. In that case, such fluorescence or luminescencemay aid in locating and/or aligning substrates.

Unless otherwise specifically stated herein, the term “microbead” isused herein as a label and does not restrict any embodiment orapplication of the present invention to certain dimensions, materialsand/or geometries.

Referring to FIG. 43, the codes that are on the beads may be indicativeof any type of desired information such as that described in U.S. patentapplication Ser. No. 10/661,082, filed Sep. 12, 2003, entitled “Methodand Apparatus for Labeling Using Diffraction Grating-Based EncodedOptical Identification Elements”. For example, in FIG. 43, the code maybe a simple code or may be a more complex code having many pieces ofinformation located in the code. In addition, the code may have checkswithin the code to ensure the code is read correctly. It can be viewedas a serial digital message, word, or frame consisting of N bits.

In particular, there may be start and stop bits 869, 871, respectively.The start and stop bits may each take up more than one bit location ifdesired. In addition, there may be an error check portion of themessage, such as a check sum or CRC (cyclic redundancy check) having apredetermined number of bits, and a code section 873 having apredetermined number of bits. The error check portion ensures that thecode which is obtained from the bead is accurate. Accordingly, having alarge number of bits in the element 8 allows for greater statisticalaccuracy in the code readout and decreases the likelihood of providingan erroneous code. Accordingly, if a code cannot be read without anerror, no code will be provided, avoiding an erroneous result. Any knowntechniques for digital error checking for single or multi-bit errors maybe used.

The code section 873 may be broken up into one or more groups of bits,for example, three bit groups 863, 865, 867, each bit group containinginformation about the bead itself or the item attached to the bead orhow the bead is to be used, or other information. For example, the firstbit group 863 may contain information regarding “identifying numbers”,such as: lot number, quality control number, model number, serialnumber, inventory control number; the second bit group 865 may contain“type” information, such as: chemical or cell type, experiment type,item type, animal type; and the third bit group 867 may contain “date”information, such as: manufactured date, experiment date, creation date,initial tracking date. Any other bit groups, number of bit groups, orsize of bit groups may be used if desired. Also, additional error orfault checking can be used if desired.

In particular, for a product manufacturing application, the code mayhave the serial number, the lot number, date of manufacture, etc. orhave other information that identifies the item and/or information aboutthe item. For a chemical or assay application, the code may haveinformation about the chemical attached to the bead, the date and/ortime of creation of the chemical or experiment, or other information ofinterest.

In addition, the digital code may be used as a covert, anti-counterfeit,and/or anti-theft type encoding, authentication, or identification code.For example, the code may contain an encrypted code that only certainpeople/entities can read and understand with the proper decryption.Also, a plurality of beads having different codes may be placed in or ona single item and all the codes would to be read together or in acertain order for them to obtain the intended tracking, identificationor authentication information. Alternatively, one of the codes may be akey to de-encrypt the codes on the other beads in the same item. Also,the codes may constantly be updated, e.g., rolling codes, or anycombination of private and/or public key encryption may be used. Anyother use of a bead combination and/or encryption/decryption techniquesmay be used if desired.

Referring to FIG. 44, the imaging properties of a known positive lens402 may be described according to the following known principles. If anobject 404 is located a distance s_(o) away from the lens 402, i.e., inan “object plane”, the lens 402 will form an image 406 in an “imageplane” of the object 404 a distance s_(i) away from the lens 402. Theknown relationship between s_(o) and s_(i) can be written as follows:

${\frac{1}{s_{o}} + \frac{1}{s_{i}}} = \frac{1}{f}$where f is the focal length of the lens 402 and s_(o) is greater thanthe focal length of the lens 402. The size of the image relative to theobject (or magnification M) has the known relationship:

$M = \frac{s_{i}}{s_{o}}$where M is the size of the image 406 divided by the size of the object404. Accordingly, if the lens 402 is placed a distance f away from theobject 404, the image 405 is infinitely large at a distance of infinityaway from the lens 402, as is known. For the purposes of thisdiscussion, the lens 402 is presumed to be infinitely large, infinitelythin (i.e, a line) as located on a plane parallel to the plane of thelens, and with no aberrations.

Referring to FIG. 45, the Fourier properties of the lens 402 may bedescribed based on the following known principles. If the lens 402 isplaced a distance f in front of an electric field distribution 408, thelens 402 will form an electric field distribution 410 that correspondsto the Fourier transform of the original electric field profile 408 at adistance f away from the lens 402 (i.e., at the “Fourier Plane” 411).The Fourier Plane image is also known as the “far field” image with adifferent scale, e.g., greater than about 20 Rayleigh ranges away. Inparticular, for the electric field sine wave 408 having a predeterminedintensity or peak value and a DC offset, resulting Fourier transformintensity pattern in the Fourier Plane 411 provided by the lens 402would be three delta functions (or points of light) 410, 412, 414,corresponding to the DC value at the point 412, the positive frequencyvalue of the sign wave 408 at the point 410 and the negative value ofthe frequency of the sign wave 408 at the point 414. The intensity ofthe light at the point 412 corresponds to the DC value of the sine wave408, and the intensity of the light at the points 410, 414 correspondsto the peak value of the sine wave 408.

Relating the Fourier Plane discussion above to the diffractiongrating-based code in the bead 8 that is read by the reader of thepresent invention, the sine wave 408 would correspond to the resultantrefractive index variation within the bead 8 having a single spatialperiod, an efficiency <100%, and where a light beam 412 is incident onthe bead at an angle of 0 degrees to the normal of the grating vector(the longitudinal axis of the bead 8).

It should be further understood from FIGS. 44,45 that if the lens 402 isplaced a distance s_(o) away from the incident electric field 408, thelens would provide an image of the electric field 408 at a distances_(i) away with a magnification s_(o)/s_(i) (not shown).

Accordingly, the reader of the present invention obtains an image of theFourier transform of the resultant refractive index variation within thebead 8, which results in lines in the Fourier plane as seen on the CCDcamera (or code camera). As a result, the reader does not requireexpensive imaging optics to obtain an image of the bead. In contrast, ifthe code on the bead could only be read by obtaining an image of thebead, e.g., if the code was simply as series of stripes printed on thebead, the reader would need to obtain a magnified image of the bead withsufficient magnification to allow a camera to read the stripes and thusobtain the code on the bead 8.

Referring to FIG. 46, the reader system is designed to minimize positionsensitivity. In particular, referring to FIG. 46, a family of curvesrepresenting the relative intensity received by a detector from auniformly fluorescent cylindrical surface illuminated by a Gaussianbeam, assuming no refraction of the beam occurs due to the bead. AllGaussian beam width data has been normalized to the bead radius and allintensity data has been normalized to the axis of the cylinder. FIG. 46Ais an expanded look at FIG. 46A in the region of interest around thenormalized signal intensity of 100%. FIG. 46, is actual data taken bymaking multiple scans on a particular bead using different axial beamposition relative to the bead. FIG. 46C is a contour plot of themultiple data sets taken for the data used in FIG. 46B, using thefollowing (approximate) beam dimensions: BeamWidth=BeadDia/4 and theBeamLength=BeadLength/2. FIG. 47 is a plot of a Gaussian beam ofwidth=bead radius, superimposed onto the bead at various offsetpositions. FIG. 48 is a plot of the relative intensity received by adetector from a uniformly fluorescent cylindrical surface illuminated byvarious width Gaussian beams passing through the axis of the cylinder.

Referring to FIG. 46, the family of curves shows the relative intensityseen by a detector system as a function of beam position relative to thebead. The reason for the shape of the curves is quite simple. For verysmall beams; as the position of the beam moves from the center of thecylinder (bead) to the edges of the bead, more surface area isilluminated, resulting in an increase in signal seen by a detector. Forvery large beams, the entire surface is essentially uniformlyilluminated and the power essentially goes as the intensity along theGaussian beam. For the case of the beam half width=the radius of thecylinder (bead), the center of the beam illuminates the center of thebead while the tails of the beam illuminate the edges of the bead (largesurface area). As the position of the beam moves from the center towardthe edges, the higher intensity light illuminates more of the surfacearea of the bead, compensating for the fact that some portion of thebeam is no longer incident on the bead and the light emitted from thebead is balanced for a relative large beam to bead position offset.These curves show that in order to maximize the offset position of thebeam to the bead, without significantly changing the amount of lightreceived by a detector, the 1/e² beam half width wants to beapproximately equal to the radius of the cylinder. Beams smaller thanthis will yield an increase in signal received at a detector as the beammoves from the axis of the cylinder. Beams larger than this will yield asignal that decreases as the beam moves from the axis of the cylinder.It is obvious to one skilled in the art that increasing the beam widthmuch larger than the cylinder diameter will also yield a signal that issubstantially insensitive to position of the beam from the axis of thecylinder. However this position insensitivity is at the expense ofrelative intensity of the signal, as well as an inability to put thebeads close together without incurring cross talk by illuminatingadjacent beads.

FIG. 46A is an expanded view of FIG. 46 in the region of interest aroundthe point where the normalized fluorescence power received by a detectoris equal to 1.

Referring to FIG. 46B, the data sets were taken by scanning a beadmultiple times along the axial length of the bead and varying thetransverse position by 2.5 microns for each successive scan. Note thatthis bead is not completely uniform in that the left side shows moresignal than the right side. FIG. 46C is a contour plot incorporating allof the data taken on the bead data shown in FIG. 46B.

Referring to FIG. 47, this family of curves represents the cylindricalshape of the fluorescent surface (circle) and a Gaussian beam of halfwidth=the cylinder radius located at different positions relative to theaxis of the cylinder. To produce the data for FIGS. 46 and 48, eachGaussian beam is plotted onto the points of the cylinder and theintensity of the points is summed.

One main difference between alternative embodiments discussed herein isthe separation of the “code” and “fluorescence” beams. This was donemainly to obtain better resolution for fluorescence while scanning thebeam parallel to the axis of symmetry of the bead, without increasingthe length of the bead. Using a beam that is larger than half of theaxial bead length causes a potential issue with reading the fluorescenceof the bead. There are two issues with this situation, adjacent beadswith widely differing surface fluorescence values and bead endconditions. Adjacent beads with widely differing fluorescence signalscan cause the fluorescence of a highly fluorescent bead to get into themeasurement of an adjacent bead with less signal. Bead end conditionscatter exists when the beads are saw cut, resulting in a surface finishthat is somewhat unknown. This can occasionally result in a bead whoseend faces have considerably more area than would be calculated by πr².Since the processes downstream put a uniform coating of materials on thesurface(s) of the beads, the ends can have more brightness than desired,or calculated. FIG. 49 represents a normalized plot of the fluorescenceof a bead scanned along the axis of the bead, with a beam approximately½ of a bead length and an end surface roughness factor of 5.

Referring to FIG. 50, the bead 8 is modeled at 225 microns long and thebeam is 128 microns long. The graph is normalized for a beam hittingonly the cylindrical portion of the bead. From the graph, it is clear tosee that there is very little of fluorescence signal from the bead thatdoes not have influence of the ends of the bead. Furthermore, it issimple to see that if two beads were touching end to end and one beadhas much more signal than the other, the ends of the bead with a lot ofsignal would influence the signal measured on the bead with very littlesignal. To reduce this effect, the bead could grow longer, the beamcould shrink, or a combination of the two.

FIG. 50 shows a calculation using the same bead and end surfaceroughness factor, but a beam approximately 3.5 times smaller than theprevious convolution, the situation is greatly improved. Now asignificant portion of the bead is free of the influence of the ends ofthe bead, therefore free from the influence of adjacent beads as well.To illustrate the effect of bead crosstalk, consider a bead between twoadjacent beads (touching end to end) with three orders of magnitude lesssignal than the beads on either side of it. This (worst case) crosstalkcan be modeled simply by multiplying the end surface factors of theprevious example by 1000. This is the FIG. 51 plot, where the case ofthe beam length equal to half the bead length (dotted line) and the casewhere beam length is one seventh the bead length (solid line) areplotted on top of one another. The differences between these two graphsillustrate the desire to use a fluorescence interrogation beam (exciter)that is 1/7th or less the axial bead length in order to have validfluorescence signal over greater than ⅓^(rd) of the bead.

Since the beam for reading code is fixed by the spacing and desiredresolution of the codes and we did not wish to change the length of thebeads, we decided it was best to use different beam diameters for codeand fluorescence in our new system.

The fluorescence pickup works in conjunction with the excitation beam toproduce signals proportional to the fluorescence of the surface of thebeads. The excitation beam comes in at an angle outside the NA of thecollection optics and excites a portion of the bead generally at thefocus of the collection optics. In doing this, it does not substantiallyilluminate unwanted material outside of our collection NA, thus keep ouroptical signal to noise ratio (OSNR) low (see page 15/16 of power pointpresentation). Furthermore, we focus the light from the collection opticinto a multimode optical fiber, which provides spatial filtering and NAfiltering for the collected signal. The optical fiber core diameter andNA are picked such that the system will collect light in the mostefficient way and make system tolerances reasonable. For the presentreader, the fiber core diameter is 100 micron and the fluorescence beamhas a diameter of about 28 micron. The fiber NA matches the collectionNA and the lens focusing light into the fiber is the same lens as thecollection optic. It is reasonable to conceive a system where the lensfocusing light into the fiber does not match the collection optics andthe fiber core diameter and NA are different from the collection optics,however as long as the product of the core diameter and fiber NA isconserved, the same collection efficiency will be achieved.

Referring to FIG. 52, alternatively, the basic architecture of aalternative reader device that can locate and read out the bead codes isshown. FIG. 53 shows a schematic of a bead mapper that is a retrofit toa conventional microscope. In this case the groove plate is madereflective so that the code beam after hitting the bead is reflectedback up into the balance of the readout optics. This allows all theoptics to be located on one side of the groove plate. FIG. 54 shows asolid model of a reader that has the laser beam incident from the bottomof the groove plate. FIG. 55 shows details of the optical path for thecase where the code laser is injected from the bottom of the plate. Twomirrors attached to a conventional microscope objective redirect thelight scattered from the bead back into the optical path. Using a pairof mirrors is advantageous in that the assembly holding both mirrors cantilt independently of the objective and will not cause a change in thebeam's angular orientation. In the figure, the readout laser is incidentfrom the bottom of the groove plate at a predeterminied angle ofincidence. The angle of incidence is about 29.7 degrees for a 532 nmreadout beam and a physical grating pitch of about 520 nm. The laserscatters from the bead and is directed via two mirrors up through a 4×objective. The objective forms an image which is subsequently Fouriertransformed onto a CCD camera, as discussed herein. FIG. 56 showsdetails of the optics used to Fourier transform the image of the beadonto a readout camera. A single spherical lens is used for this purpose.For a 4× objective, a 60 mm focal length lens can be used. The focallength of the lens determines the extent of the code “stripes” on thecamera imager.

Referring to FIG. 11, the beads 8 located on the bead holder or cell 102may be aligned on a microscope slide, e.g., a slide having lowfluorescence glass, and having grooves that hold the beads in alignment,as discussed in the aforementioned patent application. In addition, anupper glass slide may be placed on top of the grooved slide to keep anyfluid from evaporating from the plate, to minimize optical scatterduring reading.

The cell (or chamber) 102 for holding the beads may be a single cell ora sectored cell, such as that described in U.S. patent application Ser.Nos. 10/661,836 and 10/763,995 and Provisional Patent Application Nos.60/609,583 and 60/610,910, which are all incorporated herein byreference in their entirety. For example, referring to FIG. 57, a topview of a sectored cell are shown having 8 sectors. Also, referring toFIG. 58, a side view of a bead cell is shown, where beads are loadedinto the cell, moved into position by pressure waves (or “puffing”), andthen flushed out of the cell by high velocity fluid moving over thebeads, through use of ports to the device.

Alternatively, instead of using a grooved plate to align the beads 8 forreading the code, the beads may be aligned using other techniquesprovided the beads 8 are aligned property for reading. If the beads 8have a magnetic or electric polarization as discussed herein and in thecopending U.S. patent application Ser. No. 10/661,234 referenced herein,the beads 8 may be aligned using electric and/or magnetic fields. Also,a convention flow cytometer may also be used to align the beads forreading with the reader of the present invention. In that case, thebeads would flow along a flow tube and the reader would read the codeand fluorescence as the bead passes by the excitation lasers. In thatcase, the code laser and fluorescent laser may be spatially separated toallow code reading and fluorescent reading.

It should be understood that the reader need not measure bothfluorescence and codes but may just read bead codes or measurefluorescence. In that case, the components discussed herein related tothe unused function would not be needed.

Referring to FIG. 59, shows how the digital code is generated from thecamera signal. The reader provides incident light that scans along thelength of the beads and reads the codes using the code camera and opticsdiscussed herein. The codes and fluorescence may be determinedsimultaneously or sequentially. In particular, the signal from thecamera is shown as a continuous line 300 (scan data from camera). Then aseries of bit windows 302 are generated based on a start bit 304, beingthe first bit which is always a digital one. Then the peak within eachbit window 302 is determined. If the peak within a given window is abovea predetermined threshold level 306, then that bit is deemed a digitalone, if the peak is below this threshold, that bit is deemed a digitalzero. For the example in FIG. 59, a code of 41133 is shown.

FIG. 59A shows the average signal on the code camera as the plate isscanned along a groove. FIG. 60 shows the typical sequence of imagesobtained as a single bead is scanned along a groove. The peaks in theaverage signal shown in FIG. 59A correspond to the code laser scatteringoff the edge of the beads. The leading and trailing edge of the beadboth cause bright “flashes” on the code camera. These flashes are usedto locate the bead. Generally a single groove is quickly scanned forlocation of the beads. Once each bead position is known the stage canreturn to the minimum signal position between beads and read the code.FIG. 61 shows a series of beads and their corresponding holographiccodes.

Referring to FIG. 62-67, codes are read by passing a beam of light ofthe appropriate length, width and wavelength along the axis of the beadat the appropriate angle relative to the axis of the bead. This beam isdiffracted by a plurality of gratings in the core of the bead (along thecenter of the cylinder), each grating diffracting the light at adifferent angle, forming a bit within the code. This array of beams isultimately focused (imaged via Fourier transform) onto a CCD array,where the light incident on the detector array is interpreted into acode. The rate at which we can take an image of a code is a function ofthe following.

-   Scan velocity: The rate at which the beam moves past the bead.-   Bead Length: The axial length of the bead, with the scan velocity,    determine how long the beam will illuminate the code in the bead    during a scan.-   Beam Length: The length of the beam, generally along the axis of the    bead and provided it is smaller than the length of the bead,    determines what portion of the bead can be read without light    scattering onto the code area from the ends of the bead. The beam    length is further constrained by the physical size of the grating    and the required code resolution (per patent application CV-0038A,    referenced herein).-   Bead Diameter: The diameter of the bead determines how much light    will reflect off the ends of the bead, into the diffracted code    space.-   CCD Array: Detector array used to interpret codes from the    diffracted beams.-   Pixel Integration Time: The maximum pixel integration time is    bounded by the scan velocity, bead length, beam length and how much    “background” light the system can withstand without interfering with    the diffracted bits.-   Maximum Pixel Frequency: The fastest rate at which you can clock    data out of each pixel in the array determines the minimum    integration time.

Pixel Size: The physical size of the active area on each pixel (assumingthe diffracted code bits are larger than a single pixel) determines howmuch of the bit energy is captured by the array.

-   Pixel Conversion Efficiency: The efficiency for turning photons into    electrons.-   Pixel resolution: The desired resolution of the CCD array, pixels    per diffracted bit.-   Array size: will determine how small the diffracted pattern can be    focused.-   Code Intensity:-   Laser Power: Determines how much power can be delivered to the    diffraction gratings.-   Beam Width: Determines the maximum intensity that can be delivered    to the diffraction gratings.-   Bit Grating Efficiency: The efficiency at which each bit can be    diffracted from the grating.-   Code Magnification: The magnification of the code when it comes into    contact with the CCD array.-   Beam to Bead Alignment: Determines the relative position of the beam    to the bead, thus the intensity of each diffracted bit. Includes    position and angle tolerances.-   Optics Efficiency: The efficiency of the optical system between the    output of the laser and the CCD array will bound the maximum amount    of light available to read codes.

These parameters must be balanced in order to read codes from the beads.

The overall reader performance specification will drive some of theabove parameters, such as scan velocity, bead length and bead width.Some other parameters are driven by the physical limitations of ourpresent bead processing technology, such as the grating efficiency andcode resolution. If we use the same laser to read codes that is used tointerrogate fluorescence, the laser power is determined by thefluorescence specification. This only leaves a few parameters left todetermine the code detection performance. The single largest task is tofigure out the beam to bead alignment tolerances in order to determinethe minimum and maximum available code power at the CCD array. Based onthese inputs, it is a matter of choosing the CCD array with theappropriate maximum pixel frequency, pixel size, array size (which,along with the pixel resolution, determines the code magnification) andconversion efficiency to meet the code intensity requirements. Forreading the code, the beam to bead alignment has three main components;position errors generally orthogonal to the axis of the bead, in-planeangle errors (pitch) and out-of-plane angle errors (yaw). Positionerrors are reasonably straightforward in that a bead to beam offset willresult in a code generally equivalent to the intensity of the Gaussianbeam intensity at the offset position, see FIG. 62. Position errorsgenerally along the axis of the bead are not considered since alllocations along that line are scanned. The angle errors are reasonablystraightforward, if you consider the plane of incidence to contain theincident beam and a line perpendicular to the axis of the bead. Errorsabout the X axis are generally non existent since the bead is circularlysymmetric in that dimension and the system is setup to compensate forany initial angle error in that dimension. FIG. 63 shows a bead incidentby a beam with no errors. FIG. 64 shows a bead with a beam having arelative in-plane (pitch) error and FIG. 65 shows a bead with a beamhaving a relative out-of-plane (yaw) error. The system parameters fortwo different embodiments of the reader can be found in Table 1.

TABLE 1 Parameters used for different readers Parameter Embodiment 1Embodiment 2 Scan Velocity (mm/s) 30 150 Bead Length (mm) 0.45 0.225Bead Diameter (mm) 0.65 0.28 CCD: 2D array, 80 × 1D array, 1 × 256 272pixels pixels Pixel Integration Time 1 mS 0.555 mS Max Pixel Rate6528000 (readout 10000000 (pixels/sec) limited) Pixel Size (microns) 7.8× 7.8  13 × 17 Conversion Efficiency 256 bits/micro 2 joule/cm²Volts/microjoule/cm² Array size (mm) 0.624 × 2.1216 0.017 × 3.328 CodeIntensity Laser Power (mW) 10 20 Beam Width (microns) 15 28 GratingEfficiency 5.00E−04 3.00E−05 Beam to Bead Alignment: Axial Position +/−6+/−9 Tolerance (microns) In Plane angle 0.5 1.5 Tolerance (Deg) Out ofPlane angle 3 2.5 tolerance (Deg) Optics Efficiency 0.5 0.8

Referring to FIG. 66, if the trigger to find the bead edge uses the edgetrigger diode 254 discussed herein, there is a large initial peak (about20% of the laser beam), followed by the code window where the code canbe read, followed by a smaller peak for the back edge of the bead.However if the edges are jagged or random surface angles, it may bedifficult to identify the edges, especially if the beads are packed endto end. In that case, the laser power detector 243 may be used to detectthe edges of the beads, as shown in FIG. 67. The upper graph shows asingle bead and the lower graph shows 2 beads end to end. For the lowercase, a predetermined rate of change of voltage dv/dt is checked for andthen a zero crossing to identify the edge of the next adjacent bead. Inboth cases, an initial power drop below a predetermined threshold allowsdetection of the leading bead edge.

Referring to FIG. 68 an example of how the present invention would beused in an assay. First, the beads would be hybridized in solution,e.g., in a tube. Then, the beads are transferred to a reader plate (orbead holder or cell 102), and aligned in grooves. The bead cell 102 isthen placed in the reader scans along each groove, triggering when abead goes through the beam and the reader reads the code on each beadand the fluorescence of each bead, and the results are stored on adatabase in the computer. The codes and fluorescence levels may bemeasured simultaneously or sequentially. Alternatively, the beads may bein a multi-well plate and removed into the cell as discussed in pendingU.S. patent applications discussed herein. The reader may support >1200samples/hr, for <100 measurements/sample; <80 samples/hr for <1000measurements/sample; and/or about a 10 minute scan for 5,000measurements/sample.

Referring to FIGS. 69-71, show dynamic sample dynamic range data andreader throughput for the present invention. Any other specificationsmay be used depending on the application.

Although the invention has been described above as being used withmicrobeads, it should be understood by those skilled in the art that thereader maybe used with any size or shape substrate that uses thediffraction grating-based encoding techniques as described in U.S.patent application Ser. No. 10/661,234, which is incorporated herein byreference in its entirety.

The dimensions and/or geometries for any of the embodiments describedherein are merely for illustrative purposes and, as such, any otherdimensions and/or geometries may be used if desired, depending on theapplication, size, performance, manufacturing requirements, or otherfactors, in view of the teachings herein.

It should be understood that, unless stated otherwise herein, any of thefeatures, characteristics, alternatives or modifications describedregarding a particular embodiment herein may also be applied, used, orincorporated with any other embodiment described herein. Also, thedrawings herein are not drawn to scale.

Although the invention has been described and illustrated with respectto exemplary embodiments thereof, the foregoing and various otheradditions and omissions may be made therein and thereto withoutdeparting from the spirit and scope of the present invention.

1. An optical reader comprising: said reader capable of receiving atleast one encoded substrate having at least one diffraction gratingdisposed therein, said grating having a resultant refractive variationat a grating location; said grating providing an output optical signalindicative of a code when illuminated by an incident input light signalpropagating in free space; a source light providing said input lightsignal incident at a location where said substrates are located whenloaded, wherein said source light includes an optical code signal and anoptical fluorescence excitation signal; and a reader which reads saidoutput optical signal and provides a code signal indicative of saidcode.
 2. The apparatus of claim 1 wherein said output optical signalcomprises a fluorescent optical signal from said substrate and saidreader reads said output optical signal and provides a signal indicativeof said fluorescent optical signal.
 3. The apparatus of claim 1 whereinsaid optical fluorescence excitation signal comprises light having awavelength of about 633 nm.
 4. The apparatus of claim 1 wherein saidoptical fluorescence excitation signal comprises light having awavelength of about 532 nm.
 5. The apparatus of claim 1 wherein saidsubstrate is made of a glass material.
 6. The apparatus of claim 1wherein said code comprises a plurality of bits.
 7. The apparatus ofclaim 1 wherein said grating includes a number of pitches indicative ofa number of bits in said code.
 8. The apparatus of claim 1 wherein saidsubstrate has a length that is less than about 500 microns.
 9. Theapparatus of claim 1 wherein said substrate has a cylindrical shape. 10.The apparatus of claim 1 wherein said substrate is a particle or bead.11. The apparatus of claim 1 wherein said source light includes a firstlaser having said optical code signal and a second laser having saidfluorescent optical signal.
 12. The apparatus of claim 11 wherein saidfirst laser has a first beam shape and said second laser has a secondbeam shape, said first and second beam shapes being different.
 13. Theapparatus of claim 11 wherein said first laser has a first beam path andsaid second laser has a second beam path, said first and second beampaths overlapping for at least a portion of the beam paths.
 14. Theapparatus of claim 1 wherein said source light includes a first laserforming a first beam and a second laser forming a second beam, whereinthe apparatus further comprises a combiner for combining the first andsecond beams.
 15. A method of reading a code in an encoded substrate,comprising: obtaining a substrate at least a portion of which having atleast one diffraction grating disposed therein, said grating having aresultant refractive variation at a grating location; said gratingproviding an output optical signal indicative of a code when illuminatedby an incident light signal propagating in free space; illuminating saidsubstrate with said incident light signal, said substrate providing anoutput light signal, said incident light signal including an opticalcode signal and an optical fluorescence excitation signal; and readingsaid output light signal and detecting a code and fluorescencetherefrom.
 16. The method of claim 15 wherein said substrate is aparticle or bead.
 17. The method of claim 15 wherein said illuminatingsaid substrate includes using a first laser including said optical codesignal and a second laser including said fluorescent optical signal. 18.The method of claim 17 wherein said first laser has a first beam shapeand said second laser has a second beam shape, said first and secondbeam shapes being different.
 19. The method of claim 17 wherein saidfirst laser has a first beam path and said second laser has a secondbeam path, said first and second beam paths overlapping for at least aportion of the beam paths.
 20. The method of claim 17 further comprisingcombining light from said first laser and said second laser into acombined laser beam.
 21. The method of claim 15 wherein said substratehas a length that is less than about 500 microns.
 22. The method ofclaim 15 wherein said substrate has a cylindrical shape.